Percolation Theory and Compactibility of Binary Powder Systems

Defined size fractions of polyethyleneglycol powder (MW = 10,000) were mixed with defined size fractions of -lactose monohydrate in order to study the effect of compaction as a function of the weight ratios of the two excipients. For a precise control of the compression cycle, tablets were compressed on a Universal Testing Machine (Zwick 1478). Tablet tensile strength _T was quantified as a function of compressional stress _c and relative density _r using a two-parameter model with _Tmax = maximal tensile strength at zero porosity and = compressibility. The results have been analyzed on the basis of the percolation theory. As soon as the component with the lower mechanical stability is percolating the powder system, tablet hardness is controlled entirely by this component. The percolation threshold is a function of the geometrical arrangement of the particles in the compressed powder system. The expected two percolation thresholds can be distinguished as a function of the composition weight ratios if the particle size distributions of the two components differ enough.     

Antiproliferative and cytotoxic effects of some σ<Subscript>2</Subscript> agonists and σ<Subscript>1</Subscript> antagonists in tumour cell lines

To establish the activity of ligands at ₁ and ₂ receptor, we chose two tumour cell lines, the human SK-N-SH neuroblastoma and the rat C6 glioma lines, which express ₂ receptors at a high density and ₁ receptors in their high-affinity or low-affinity state. We tested the ₂ receptor agonist PB28 and the ₂ antagonist AC927, and (+)-pentazocine and NE100 as agonist and antagonist, respectively, at ₁ receptors, with regard to antiproliferative and cytotoxic effects. In addition, 1,3-di(2-tolyl)guanidine (DTG) and haloperidol were tested as reference compounds displaying nearly equipotent affinity (₂>₁ and ₁>₂, respectively). In both SK-N-SH and C6 cells, PB28 and NE100 displayed the most potent results both in antiproliferative and cytotoxic assay while AC927 and (+)-pentazocine were inactive in both assays. The cytotoxic and antiproliferative effects of DTG and haloperidol reflected their ₁ antagonist activity and ₂ agonist activity. Moreover, our results in the tumour cell lines correlated well with those for ₂ activity found previously in a functional assay in the guinea-pig bladder. These findings establish a new model for evaluating both ₂ and ₁ receptor activity of ligands, which could be useful for developing new ligands having mixed ₂ agonist/₁ antagonist activity as potential antineoplastic agents.     

A new method for evaluating σ<Subscript>2</Subscript> ligand activity in the isolated guinea-pig bladder

We demonstrated the presence of ₂ receptors in the guinea-pig ileum by saturation analysis and extended our investigation to guinea-pig bladder and rat bladder. In functional assays of the isolated tissues in organ baths, ₂ receptor ligands inhibited electrically evoked contractions in both guinea-pig bladder and ileum and a linear correlation was found between ₂ receptor affinity and ₂ receptor activity values of selected compounds. The ₂ activity of these compounds in the presence of desensitised ₁ receptors both in bladder and ileum was also tested. On the basis of our results, we propose the electrically stimulated guinea-pig bladder as a new method for evaluating ₂ activity.     

Frequency- and train length-dependent variation in the roles of postjunctional α1- and α2-adrenoceptors for the field stimulation-induced neurogenic contraction of rat tail artery

Summary The present paper examines the roles of postjunctional ₁- and ₂-adrenoceptors for the noradrenaline (NA)-induced neurogenic contractile response to field stimulation mainly with 1–100 pulses at 2 or 20 Hz, in the tail artery of adult normotensive rats. Pharmacological tools were employed to isolate and characterize the ₁- and ₂-adrenoceptor-mediated components of this response. The degree to which the drugs influenced NA release or reuptake was assessed by their effects on the electrochemically determined, stimulation-induced rise in the NA concentration at the innervated outer surface of the media. This response was unaffected by ,-methylene ATP (10 M) or suramin (500 M), added to desensitize or block P₂-purinoceptors, respectively prazosin (0.1 M) or SK&amp;F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxyl]-3-methyl-1H-2, 3, 4, 5-tetrohydro-3-benzazepine, 0.1 M), used to block postjunctional ₁- and ₂-adrenoceptors respectively, nifedipine (10 M), blocker of Ca²⁺ influx through L-type channels, and ryanodine (10 M), which blocks mobilization of Ca²⁺ from intracellular stores; it was moderately enhanced by yohimbine (0.1 M), blocker of pre- and postjunctional ₂-adrenoceptors, and strongly enhanced by cocaine (3 M) or desipramine (1 M), blockers of NA reuptake. Judging from their inhibitory effects on the contractile responses to the ₁- and ₂-adrenoceptor agonists, phenylephrine andxylazine, prazosin (0.1 M)and SK & F 104078 (0.1 M) could be used to selectively block ₁- and ₂-adrenoceptors respectively, while yohimbine (0.1 M) was less selective, strongly depressing ₂- and slightly depressing ₁-adrenoceptor-mediated responses. The ₁-adrenoceptor-mediated component of the contractile response to short trains at 20 Hz was fast in onset, brief in duration and abolished by ryanodine; that mediated by ₂-adrenoceptors was more delayed, prolonged and insensitive to ryanodine. Both components were dose-dependently depressed by nifedipine (0.1–10 M). The small contractile responses to single pulses, or up to 50 pulses at 2 Hz, or short train (< 4 pulses) at 20 Hz, were more markedly depressed by 0.1 M yohimbine or SK & F 104078 than by 0.1 M prazosin and, hence, mediated mainly by ₂-adrenoceptors. The reverse was true of the much larger response to longer trains at 20 Hz, which thus probably was mediated mainly by ₁-adrenoceptors. Cocaine or desipramine, as well as ,-methylene ATP or suramin, amplified both components of the NA induced contractile response especially that mediated via a₁-adrenoceptors and caused by single pulses or short trains.The main conclusions are (i) that the small NA-induced contractile responses of this artery to single pulses, or pulses at low frequency, or in short trains at high frequency, are mediated mainly via ₂-, and the larger responses to longer trains at high frequency increasingly via ₁-adrenoceptors, (ii) that the ₁- and ₂-adrenoceptor-mediated components interact cooperatively, probably at least in part by utilizing two different pathways to increase the intracellular Ca²⁺, (iii) that neuronal reuptake of NA strongly restricts both components of the NA-induced contraction, especially the ₁-adrenoceptor-mediated response to single pulses or short trains, and (iv) that both components of the NA-induced contraction, especially that mediated by ₁-adrenoceptors, may be depressed by ATP released by field stimulation and acting via P_2x-purinoceptors on smooth muscle. Based on these results a novel working hypothesis is proposed, in which it is assumed that the geometry of NA-mediated neuromuscular transmission in this vessel varies with the frequency and number of impulses in a stimulus train.Correspondence to J.-X. Bao at the above address     

Cosolvency and Cosolvent Polarity

The solubilities of three poorly soluble drugs, phenytoin, benzocaine, and diazepam, in cosolvent– water mixtures have been previously shown to be approximated by the log-linear solubility equation; log (S _m/S _w) = f, where S _m and S _w represent the solubilities of the drug in the solvent mixture and water, respectively, f is the volume fraction of cosolvent, and is the slope of a plot of log (S _m/S _w) vs f. In this study, the slopes, , of the solubility plots were related to indexes of cosolvent polarity including the dielectric constant, solubility parameter, surface tension, interfacial tension, and octanol–water partition coefficient. Those polarity indexes that reflect the cohesive properties of the solvents such as the solubility parameter and interfacial tension resulted in the highest correlations with the slope, . The hydrogen bonding ability of the neat cosolvent, expressed as the density of proton donating groups (HBD) or acceptor groups (HBA), was also found to be highly correlated with . Additional relationships derived from theories involving solubility parameters and interfacial tension provide improved correlations between the cosolvent polarity and . These results and analysis provide the basis for the estimation from physicochemical parameters of the appropriate type and amount of cosolvent needed to solubilize nonpolar drugs.     

Biphasic effects of sigma ligands on the neuronal response to N-methyl-D-aspartate

Previous studies from our laboratory have demonstrated that low doses of selective sigma () ligands potentiate the neuronal response to N-methyl-D-aspartate (NMDA) in the CA₃ region of the rat dorsal hippocampus. Sertraline and clorgyline, two antidepressant drugs with a high affinity for receptors, also potentiate, at low doses, the NMDA response; however, when administered at higher doses, the degree of potentiation induced by these two ligands progressively decreases (Bergeron et al. 1993). In the present experiments, the selective ligands DTG, ( + )pentazocine, BD-737, JO-1784 and L-687,384 were studied to determine if they would also generate bell-shaped dose-response curves. These ligands were administered intravenously at doses ranging from 1 g/kg to 1 g/kg or applied by microiontophoresis. They potentiated selectively, with bell-shaped dose-response curves, the NMDA-induced activation of pyramidal neurons in the CA3 region of the rat dorsal hippocampus. The potentiation of the NMDA response following the intravenous administration of a low dose of a ligand persisted for at least 60 min, after which point in time a second injection of the same dose induced the same degree of potentiation. Moreover, a sustained potentiation was obtained during prolonged microiontophoretic applications of a ligand. These two latter series of observations suggest that the lack of effect of the high doses of 6 ligands is not related to a rapid desensitisation of receptors. This biphasic effect of ligands might be due to the concomitant actions of these ligands on distinct subtypes of receptors.     

On the Variability of Dissolution Data

Purpose. To investigate dissolution data variability and its origins. Methods. The Weibull function with four parameters, t₀ (dissolution lag-time), K (the rate parameter), (the shape parameter) and D (the fraction dissolved as t ), is used to describe the dissolution curve. The variance of the dissolution data is expressed in terms of these parameters and their individual variances ² _t0, ² _K;, ² , and ² _D. These four variances originate from variable physical properties of the dosage units and from a variable dissolution environment. Therefore, dissolution data variability depends on both, the functional form of the curve and on the variance of the physical conditions. The use of this method enables the elucidation of the sources of dissolution data variability. Results. In the case of a sigmoidal dissolution curve ( > 1), data variance is zero as dissolution begins (following dissolution lag-time). This initial variance diverges when the dissolution curve is non-sigmoidal (with < 1) but assumes a finite value, proportional to the dissolution lag-time variance (² _t0) when the data fits a regular first order rate curve ( = 1). Following a long dissolution time, data variance attains a constant value equal to the dissolution extent variance, ² _D. When the dissolution curve is sigmoidal and the variability related to the dissolution extent is sufficiently small (_D/D << 1), a maximum in the variance is expected at some intermediate time point (corresponding to the curve inflection point, when the main source of variability is dissolution lag-time t₀, or around t = 1/K + t₀, when the main sources of variability are the rate parameter K or the shape parameter ). When the curve is sigmoidal ( > 1) and the main source of variability relates to the dissolution extent, the overall variance grows with time all the way to the plateau of the dissolution curve. With a non-sigmoidal dissolution curve ( 1), data variability decreases with time soon after dissolution begins. In that case, if the main source of variability is the dissolution lag-time (t₀), the variance decreases all the way to the plateau of the dissolution curve. If the dissolution extent, D, is the main source of variability, a minimum in the variance is expected at some intermediate time point. The dissolution relative variance, on the other hand, diverges as dissolution begins and decreases with time at least until 63% of the drug is released, irrespective to the Weibull parameter values. Later, it may decrease or increase, attaining a fixed value (² _D/D²) at the plateau of the dissolution curve. Conclusions. The particular time dependence of dissolution data variance is well defined in terms of the Weibull shape parameters and their individual variances. Dissolution data variability may decrease or increase with time along the curve. It may attain a maximum or a minimum value at some intermediate time point. It may converge or diverge as dissolution begins. When the dissolution data is well fitted to the Weibull function, the sources of data variability (in terms of the Weibull parameters) may be elucidated. The variability of dissolution data originates from physical sources but is also dependent on the functional form of the curve.     

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

Summary Presynaptic ₂-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [³H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The ₂-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC₅₀ values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant K_D. The K_D values correlated only weakly between the rat and the rabbit. Dissociation constants K_A of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the ₂-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The K_A value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic ₂-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the _2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the _2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of ₂-RG20, the putative rat _2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of ₂-C10, the putative human _2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the ₂-C10 (r = 0.98; P<0.001) but not with the ₂-RG20 gene product. Since presynaptic ₂-autoreceptors are also _2D in rat submaxillary gland and perhaps vas deferens and _2A in rabbit pulmonary artery, the possibility arises that the majority of ₂-autoreceptors generally are _2D in the rat and _2A in the rabbit. Moreover, receptors of the _2A/D group generally may be the main mammalian ₂-autoreceptors.Correspondence to: N. Limberger at the above address     

The sigma<Subscript>1</Subscript> (σ<Subscript>1</Subscript>) receptor activation is a key step for the reactivation of cocaine conditioned place preference by drug priming

Rationale Cocaine-seeking behavior can be investigated in rodents using the conditioned place preference (CPP) paradigm, in which the drug-paired environment serves as a conditioned stimulus. Such approach allowed to previously demonstrate the importance of the neuromodulatory sigma₁ (₁) receptor in acquisition of cocaine-induced CPP. CPP can be extinguished and then reactivated, notably using a cocaine challenge (i.e., priming).Objectives and methods In order to examine the role of the ₁ receptor in reinstatement of Cocaine-seeking, Swiss mice acquired CPP with cocaine (30 mg/kg, ip) and then CPP was extinguished.Results A challenge cocaine priming (15 mg/kg) reactivated CPP up to 140% of the post-conditioning response. Pre-administration of the ₁ receptor antagonist BD1047 (330 mg/kg, ip) or repeated treatment with an antisense probe targeting the ₁ receptor prevented CPP reactivation. The ₁ agonist igmesine (1–10 mg/kg, ip) or the steroid dehydroepiandrosterone (DHEA, 10–40 mg/kg, sc) reactivated CPP, in a BD1047-sensitive manner. Moreover, the in vivo [³H](+)-SKF-10,047 binding levels to the ₁ receptor were increased after cocaine conditioning in numerous brain structures and these increases subsisted after extinction. Finally, cross-reactivation of cocaine-induced CPP was observed after phencyclidine (PCP), morphine, nicotine and ethanol administration. However, BD1047 blocked reactivation of CPP induced by PCP, morphine and nicotine but not ethanol.Conclusions Since activation of the ₁ receptor is not sufficient to sustain CPP in naive animals [Neuropsychopharmacology 26 (2002) 444], it is concluded that ₁ receptor activation is a key event for relapse to drug seeking. Activation may occur via sensitization due to enhanced in vivo available of receptors.     

Simple model to explain effects of plasma protein binding and tissue binding on calculated volumes of distribution,apparent elimination rate constants and clearances

Summary A simple pharmacokinetic model, incorporating linear plasma protein binding, linear tissue binding, and first order elimination of free (unbound) drug, was studied. If Cl_p is the plasma clearance, V_f is the true volume of distribution of free drug, is the apparent elimination rate constant, is the fraction of the drug which is free in plasma, f is the fraction of the drug which is free in the entire body, k_f is the intrinsic elimination rate constant for free drug, and A _TB ^o is the initial amount of drug which is bound to tissues, then the model indicates that the following relationships hold: (1) Cl_p = V_f k_f; (2) = f k_f; and V_dext = (/f) V_f. Only , and not f, can be measuredexperimentally. Dividing Cl_p by provides an estimate of the intrinsic clearance of free drug, V_fk_f. A plot of V_dext versus has an intercept equal to V_f, and the ratio of the slope/intercept is an estimate of A _TB ^o /A _f ^o , where A _f ^o is the initial amount of free drug (equal to V_f times initial concentration of free drug in plasma). Thus, an estimate of A _TB ^o may be obtained. Dividing the intrinsic clearance by V_f provides an estimate of k_f. Thus, theoretically, estimates of V_f, k_f, A _TB ^o and f may be obtained. The variables are not separated when is plottedversus , and curvature of such plots is expected; no useful information is obtained from such plots.Partly supported by Public Health Service Grant 5-P-11-GM 1559 and partly by Grant 1RO1AAOO683-O1A1 from the National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland.     

Blockade of γ-aminobutyric acid-receptors of the B-subtype inhibits the dopamine-induced enhancement of the release of cholecystokinin-like immunoreactivity from slices of rat dorsal caudatoputamen

Summary When slices of rat dorsal caudatoputamen (= neostriatum) are incubated in vitro, Choecystokinin-like immunoreactivity (CCK-LI) is released upon addition of veratridine (3.75 mol/l). This release is affected by dopamine and by -aminobutyric acid (GABA)-receptor agonists. Dopamine enhances the release by stimulating dopamine D₂-receptors and decreases it via D₁-receptors. GABA_A-receptor agonists enhance the veratridine-induced release of CCK-LI, while GABA_B-receptor agonists decrease it. In the present investigation, it was examined whether GABA-receptors are involved in the effect which dopamine exerts via D₂-receptors. The GABA_A-receptor antagonist bicuculline (10 mol/l)and the blocker of the GABA_A-receptor ionophore picrotoxin (1 mol/l) did not affect the dopamine (0.1 mol/1)-induced increase in the release of CCK-LI. However, the GABA_A-receptor agonist muscimol (1 mol/l) not only enhanced the release of CCK-LI, but also prevented a further enhancement by dopamine (0.1 mol/l). This effect of muscimol was blocked by bicuculline (10 mol/l). In the presence of -amino-n-valeric acid (0.1 mmol/l), which has been described to block GABA_B-receptors, dopamine no longer enhanced the veratridine-induced release of CCK-LI. -Amino-n-valeric acid also inhibited the pronounced enhancement of the release of CCK-LI caused by dopamine (0.1 mol/l) and 1 mol/l in the presence of the preferential D₁-receptor antagonist SCH 23390. The effect of -amino-n-valeric acid persisted in the presence of bicuculline (10 mol/l and 100 mol/l). (+)-Baclofen, a partial agonist at GABA_B-receptors, and the stereoisomer (–)-baclofen, a full agonist, also prevented the effect of dopamine on the veratridine-induced release of CCK-LI. The effects of both drugs may be due to desensitization of GABA_B-receptors, which has been described to develop quite rapidly. It is concluded that -amino-n-valeric acid blocks GABA_B-receptors and in this way prevents the enhancement of the veratridine-induced release of CCK-LI caused by dopamine via D₂-receptors. These data are interpreted as evidence that dopamine and GABA-neurons can directly or indirectly interact in the rat neostriatum. Send offprint requests to D. K. Meyer at the above address     

Possible subdivision of postsynaptic α-adrenoceptors mediating pressor responses in the pithed rat

Summary Additional evidence has been obtained indicating a possible subclassification of postsynaptic -adrenoceptors into ₁ and ₂-subtypes. The pressor responses to the -adrenoceptor agonists L-phenylephrine and guanfacine were quantified after i.v. administration to pithed rats. The -sympatholytic drug yohimbine (1 mg/kg) displaced both dose-response curves to the right, but the effect was greatest for guanfacine. After prazosin (0.1 mg/kg) a 53-fold shift to the right was noticed for the dose-response characteristic of L-phenylephrine. Prazosin antagonized the effect of only the higher doses of guanfacine. The findings indicate that L-phenylephrine and prazosin preferentially interact with ₁-adrenoceptors as agonist and antagonist, respectively. Yohimbine proved less selective than prazosin, but preferentially blocks postjunctional ₂-adrenoceptors in the vascular wall. The results obtained with guanfacine may be interpreted to indicate that this drug acts on ₂-adrenoceptors at lower doses and additionally stimulates ₁-adrenoceptors at higher ones. Preliminary findings with corynanthine and rauwolscine support this interpretation.     

Model-independent assessment of accumulation kinetics based on moments of drug disposition curves

Summary The bounds of the accumulation profile can be predicted on the basis of the mean disposition residence time (MDRT) of a drug. The time to reach 90% of the plateau level (t _0.9) is less than 3.7 MDRT. This prediction can be improved if, in addition, the variance of disposition residence time (VDRT, CV _D ² =VDRT/MDRT²), or the terminal exponential coefficient (), is known. For CV _D ² 1 or MDRT1, the time to reach steady state (t_0.9) approaches 2.3 MDRT (limiting case of monoexponential drug disposition curve). Conditions are stated under which can be regarded as the principal determinant of the accumulation rate.     

Regulation of adenylate cyclase activity in hamster adipocytes

Summary In ghosts of hamster adipocytes, the regulation of adenylate cyclase (ATP: pyrophosphate lyase, cyclizing; EC 4.6.1.1) activity by prostaglandins, -adrenergic agonists and nicotinic acid was studied. These three classes of antilipolytic agents caused adenylate cyclase inhibition without an apparent lag phase. Maximal inhibitions observed ranged between about 45% (by -adrenergic agonists) and 60% (by prostaglandins and nicotinic acid). The order of potency for the inhibitory prostaglandins (PG) was PGE₁ PGE₂>PGF₂PGI₂>PGD₂>6-keto PGF₁. The IC₅₀ values obtained were about 0.007, 0.06, 0.3 and 1 M for PGE₁, PGF₂, PGD₂ and 6-keto PGF₁, respectively. -Adrenergic agonists, studied in the presence of the -adrenergic blocking agent, propranolol (30 M), inhibited the fat cell enzyme with the order of potency (1)-adrenaline > (1)--methylnoradrenaline (1)-noradrenaline > clonidine tetryzoline > (1)-phenylephrine. The IC₅₀ values obtained for (1)-adrenaline and (1)-noradrenaline were about 3 and 10 M, respectively. The inhibitory effect of (1)-adrenaline was blocked by the -adrenergic antagonists with the potency order yohimbine phentolamine > prazosin. These findings suggest that an ₂ of receptors is involved in this catecholamine-induced inhibition. Nicotinic acid (10 M) reduced adenylate cyclase activity by about 60% with half-maximal effectiveness at about 0.6 M. The nicotinic acid derivatives, nicotinamide, -pyridylcarbinol and NAD (up to 100 M), had no effect on enzyme activity.Inhibition of the hamster adipocyte adenylate cyclase by the antilipolytic agents required the presence of both GTP, which reduced basal activity by about 80% at 10 M, and sodium ions, which specifically activated the GTP-affected from of the enzyme. Inhibition was also observed in the presence of ACTH, which in a GTP-dependent manner increased adenylate cyclase activity. Pretreatment of the enzyme preparation with NaF (10 mM) partially reduced the inhibitory effect, and preactivation with the stable GTP analogue, guanylyl 5-imidodiphosphate (100 M), abolished the adenylate cyclase inhibition by the antilipolytic agents.Abbreviations PG prostaglandin - GMP-P(NH)P guanylyl 5-imidodiphosphate Some of the data were presented in abstract form (Aktories et al., 1979a)     

α2-autoreceptors and α2-heteroreceptors modulating tyrosine and tryptophan hydroxylase activity in the rat brain in vivo: an investigation into the α2-adrenoceptor subtypes

The subtype determination of auto- and hetero-₂-adrenoceptors modulating the synthesis of noradrenaline (NA) and serotonin (5-HT), respectively, was assessed using the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition as a measure of the rate of tyrosine and tryptophan hydroxylation in the rat brain in vivo.In the cerebral cortex and hippocampus, Org 3770 (non-selective a2-adrenoceptor antagonist, 0.5–10 mg/kg, i.p.) increased (43%–58%) and clonidine (non-selective ₂-adrenoceptor agonist, 1 mg/kg) decreased (37%–49%) the synthesis of dopa. Also the antagonist ARC 239 (_2B/C selective, 5–40 mg/kg) increased the synthesis of dopa in cortex (39%–46%) and hippocampus (17%–85%). In contrast, the antagonist BRL 44408 (_2D selective, 1–10 mg/kg) did not increase the synthesis of dopa in cortex, and increased it modestly in hippocampus only. The agonist guanoxabenz (_2B/C selective, 0.03–3 mg/kg) decreased the synthesis of dopa in both brain regions (20%–65%), whereas the agonist oxymetazoline (_2D selective, 0.1–3 mg/kg) failed to do so. These results indicated that the ₂-autoreceptors that modulate the synthesis of dopa/NA are probably associated with the _2B/C-subtypes.In cortex and hippocampus, clonidine decreased (35%–53%) the synthesis of 5-HTP but Org 3770 failed to induce the opposite effect (except the 2 mg/kg dose in cortex). BRL 44408 markedly increased the synthesis of 5-HTP in cortex (113%–148%) but not in hippocampus. Similarly, also ARC 239 increased the formation of 5-HTP in cortex (36%–48%) but not in hippocampus, where it was decreased (30%–55%). Oxymetazoline decreased the synthesis of 5-HTP in hippocampus (28%–30%) but failed to do so in cortex. Guanoxabenz in the low dose range (0.03–0.3 mg/kg) did not decrease the synthesis of 5-HTP in any brain region. These results indicated that the ₂-heteroreceptors that modulate the synthesis of 5-HTP/5-HT may well be different from the proposed _2B/C-autoreceptors modulating the synthesis of dopa/NA. These ₂-heteroreceptors appear to be associated with the _2D-subtype.     

In vivo electrophysiological evidence for tonic activation by endogenous noradrenaline of α2-adrenoceptors on 5-hydroxytryptamine terminals in the rat hippocampus

Summary The activation of ₂-adrenergic heteroreceptors was studied by comparing the effectiveness of the electrical stimulation of the ascending 5-HT pathway in suppressing the firing activity of CA₃ dorsal hippocampus pyramidal neurons prior to, and following, the intravenous administration of noradrenergic agents. Desipramine (2 mg/kg), a selective noradrenaline reuptake blocker, reduced the efficacy of the stimulation; this effect was reversed by the ₂-adrenoceptor antagonists yohimbine (0.5 mg/kg) and (-)mianserin (0.5 mg/kg), but not by idazoxan (0.5 mg/kg), an adrenoceptor antagonist with preferential affinity for the imidazoline recognition sites. Low doses of the ₂-adrenoceptor agonist clonidine (2 and 10 g/kg) enhanced the efficacy of the stimulation, while high doses (100 and 400 g/kg) reduced it. These incremental and decremental effects of clonidine were reversed by 0.1 and 1 mg/kg of yohimbine, respectively. The enhancing effect of the low dose of clonidine (10 g/kg) was abolished in rats pretreated with the noradrenaline neurotoxin 6-hydroxydopamine. However, the inhibitory effect of a high dose of clonidine (100 g/kg) was unaltered by this pretreatment. These results indicate that low doses of clonidine preferentially activate ₂-adrenergic autoreceptors on the noradrenaline neurons resulting in a reduction of the tonic inhibitory effect of endogenous noradrenaline on 5-HT neurotransmission, while higher doses of clonidine would decrease 5-HT neurotransmission through the direct activation of ₂-adrenergic heteroreceptors on 5-HT terminals. Furthermore, the selective ₂-adrenergic heteroreceptors antagonist(-)mianserin (0.5 mg/kg) increased by itself the efficacy of 5-HT neurotransmission, an effect not observed with yohimbine and idazoxan. Taken together, these results suggest that, in vivo, the ₂-adrenoceptors on 5-HT terminals of the rat hippocampus are tonically activated by endogenous noradrenaline and modulate 5-HT release. Correspondence to R. Mongeau at the above address     

Distinct distribution of opioid receptor types in rat lumbar spinal cord

Summary The distribution of opiate binding sites was studied in sections of rat lumbar spinal cord under conditions selective for , and receptors. While the levels of binding sites were highest in the substantia gelatinosa, elevated levels were also observed in laminae III, IV, V and VIII. In contrast, binding was notable only in lamina I. The levels of typical sites were low, and were concentrated in the substantia gelatinosa. An additional, atypical site was detected using ³H-diprenorphine in the presence of , and receptor blocking agents, and this site was also concentrated in the substantia gelatinosa. Send offprint requests to B. J. Morris at the above address     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Über die Wirkung von α-Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen

Zusammenfassung 1. Versuche über die Wirkung von -Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen haben ergeben, daß Noradrenalin im Herzen, sowie Dopamin und Noradrenalin im Gehirn durch äquimolare Mengen von -Methyl-Noradrenalin bzw. -Methyl-Dopamin ersetzt werden.2. Die Adrenalinverarmung der Nebennieren nach Vorbehandlung mit -Methyl-Dopa wird demgegenüber nur zu einem geringen Teil durch die methylierten Brenzcatechinamine -Methyl-Noradrenalin und -Methyl-Dopamin ausgeglichen. Ein weiteres Brenzcatechinamin konnte nicht nachgewiesen werden, so daß -Methyl-Adrenalin wahrscheinlich nicht gebildet wird.3. Im Meerschweinchenherzen gespeichertes -Methyl-Noradrenalin wird durch Reserpin in vivo schlechter freigesetzt als Noradrenalin.4. Das in isolierten Herzgranula (Sediment 100 000·g) gespeicherte -Methyl-Noradrenalin wird durch Segontin bei der Inkubation in vitro ebenfall schlechter freigesetzt als Noradrenalin aus normalen Herzgranula.5. Der Mechanismus der -Methyl-Dopa-Wirkung wird diskutiert.6. Es wird eine fluorimetrische Methode zur Bestimmung von -Methyl-Noradrenalin angegeben.

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Summary 1. The catecholamine content of guinea pig organs after administration of -methyldopa has been determined. The results show that the loss of noradrenaline from heart and that of noradrenaline and dopamine from brain is compensated by a stoichiometric uptake of -methylnoradrenaline and -methyldopamine respectively.2. On the contrary the loss of adrenaline from the suprarenal medulla induced by pretreatment with -methyldopa is not completely restored by the -methylated amines, -methyldopamine and -methylnoradrenaline. -methyladrenaline could not be detected even by paperchromatography.3. The release of the stored -methylnoradrenaline from guinea pig heart by reserpine is very small in comparison with that of noradrenaline.4. Furthermore only small amounts of the stored -methyl-noradrenaline are released from isolated granules of the guinea pig heart after incubation in vitro with segontin. Whereas the same amount of segontin depleted completely the noradrenaline content of the granules isolated from normal hearts.5. The mechanism of action of -methyldopa is discussed.6. A method for the fluorimetric determination of -methylnoradrenaline is described. It is at first separated from -methyldopamine, dopamine and histamine by column chromatography, condensed with o-phthaldialdehyd and the obtained fluorescense is measured by using an Aminco-Bowman spectrophotofluorimeter.

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Mit 6 Textabbildungen

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Herrn Dr. H. Blaschko zum 65. Geburtstag gewidmet.

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Über einen Teil der Ergebnisse wurde auf der Frühjahrstagung der Deutschen Pharmakologischen Ges. in Mainz, April 1964, berichtet. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 247, 297 (1964).

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Clonidine inhibits salivary secretion by activation of postsynaptic α2-receptors

Summary The effects of clonidine on the submaxillary gland of the rat were studied. Doses ranging between 100 to 3.000 g/kg produced a sustained secretory response which was blocked by 0.1 mg/kg of prazosin but not by 1 mg/kg of yohimbine. Clonidine 10 g/kg markedly inhibited the salivation induced by noradrenaline, methacholine and substance P but not that induced by isoproterenol. The inhibition caused by the ₂-agonist was greater for noradrenaline than for either methacholine or substance P. Blockade of ₂ adrenoceptors with yohimbine (0.3–1 mg/kg) prevented the inhibition by clonidine of noradrenaline, methacholine and substance P induced salivation. On the other hand, prazosin 0.1 mg/kg did not modify the inhibition by clonidine of methacholine induced secretion. The results obtained indicate that clonidine exerts a dual effect on salivary secretion: at high doses it elicits salivation through activation of ₁-adrenoreceptors; at the dose of 10 g/kg clonidine activates ₂-adrenoreceptors which inhibit the secretory response evoked through either muscarine, substance P and ₁-adrenorecptors agonists.Partially supported by grants No 3111 j/82, CONICET and 20241/82-9, SUBCYT